MicroRNAs are dysregulated in nearly all cancers. By inhibiting oncogenic or tumor suppressive messenger RNAs, miRNAs can have a significant effect on cancer development and pro- gression. There is thus great interest in modulating the levels of various miRNA species for the treatment of cancer. Among the most promising molecules for inhibiting oncogenic miRNAs (oncomiRs) are artificial anti-sense oligonucleotides known as peptide nucleic acids (PNAs). However, their delivery into tumor cells is a challenge, as they are relatively cell-impermeable. This challenge can be overcome via the use of pH (low) in- sertion peptide (pHLIP), a short helical peptide capable of inserting across a cell membrane to form a transmembrane helix at acidic pHs, but not at normal physiologic pH. At acidic pHs, PNAs attached as cargo molecules to the inserting end of pHLIP are delivered across the cell membrane and into the cytosol. Since tumors are acidic, the PNAs accumulate preferentially in tumors, thereby limiting off-target effects. Recently, proof-of-concept therapeutic efficacy of the pHLIP-PNA technology was established in a mouse model of 'on- comiR-addicted' lymphoma, which undergoes complete regression upon withdrawal of the oncomiR. However, as oncomiR addiction has yet to be observed in nature, this model is of limited clinical relevance. We hypothe- size that the pHLIP-PNA technology will also be effective in other models with different oncomiRs, and the technology can be enhanced by the use of short G-clamp gamma-modified PNAs (G-MP-?-PNAs), which are smaller than the original PNAs but exhibit similar binding affinity and increased solubility. Specific Aims: This proposal is comprised of three specific aims The first aim is to demonstrate more robust therapeutic relevance while broadening the scope of the pHLIP-PNA technology to inhibit oncomiRs in other cancers, which would prove valuable for translating the technology to the clinic. To do this, we will demonstrate therapeutic efficacy in the highly robust and clinically relevant Braf/Pten mouse model for malignant melanoma. The second aim is to investigate the hypothesis that the use of short G-MP-?-PNAs, due to their enhanced solubility and smaller size, will result in increased tumor-specificity and delivery of th pHLIP-PNA technology, thereby increasing the therapeutic efficacy. Lastly, the third aim of the proposal is to elucidate why certain miRNAs are oncogenic in some cancers but tumor suppressive in others, which is of critical importance to the development of oncomiR- inhibiting therapies. As an individual miRNA can target tens to hundreds of different mRNA molecules, some of which may be oncogenic while others are tumor suppressive, we hypothesize that it is the balance between the miRNA's oncogenic and tumor suppressive targets which determines whether the miRNA will have a net onco- genic or net tumor suppressive effect. A computational model we developed predicts this, and we now aim to provide experimental support for its predictions by switching a miRNA from oncogenic to tumor suppressive and vice versa in cell lines by modifying the expression level of one of the miRNA's oncogenic targets.